A variety of techniques have been developed to analyze nucleic acids, proteins (e.g. receptors), and other biological analytes for the presence of interactions, mutations, or other characteristics of interests. Such techniques can be used to determine, for example, if a patient has a particular disease or has a predisposition toward the disease. That is, nucleic acid-based analysis can be used to verify the presence or absence of expressed genes or polymorphisms. Nucleic acid-based and other analysis can also be used to monitor progression of disease, assess effectiveness of therapy or to modify dosage formulations. Protein binding assays can be used to test for specific proteins in blood or cell extracts. Antibody binding assays can be used to detect a number of analytes including small molecules and proteins.
One technique for analyzing biological analytes employs a microarray (or microelectronics biochip) that generates a hybridization pattern representative of binding characteristics of a target analyte within the sample or a pattern of binding events on a protein or antibody array (see, e.g., Schena (ed.), (2000) Microarray Biochip Technology, Eaton Publishing, Natick, Mass.; Vrana et al., (2001) “Microarrays and Related Technologies: Miniaturization and Acceleration of Genomic Research”, Cambridge Healthtech Institute Report 8, May 2001). In one example, a nucleic acid microarray can include a rectangular array of immobilized single stranded nucleic acid fragments. Each element within the array includes a few tens to millions of copies of identical single stranded strips of nucleic acid containing specific sequences of nucleotide bases. Identical or different fragments of a nucleic acid can be provided at each different element of the array. For example, in a rectangular microarray, location (1,1) can contain a different single stranded fragment of a nucleic acid than location (1,2), which can also differ from location (1,3), and so on. See, e.g., Schena (ed.), (2000) Microarray Biochip Technology, Eaton Publishing, Natick, Mass.; Pirrung, (1997) Chem. Rev. 97: 473-486.
Generally, microarrays typically employ fluorescence or electrical phenomenology to indicate a positive detectable result. In one method that employs fluorescence imaging, a double stranded nucleic acid sample to be analyzed is first separated into individual single stranded sequences and then fragmented into smaller probes. Alternatively, single stranded sequences can be synthesized in a DNA synthesizer. Each probe is then tagged with a fluorescent molecule. The probes are applied to the microarray, at which point each probe binds only with complementary nucleic acid fragments embedded on the microarray. Probes that are not complementary to any of the elements of the microarray will not bind to the microarray and can be discarded during subsequent fluidic reactions and washes. Thus, only those nucleic acid samples in the microarray that contain fragments that bind complementary sequences of the probe nucleic acid sample will hybridize with probes containing fluorescent molecules. Typically, a fluorescent light source is then applied to the microarray to generate a fluorescent image identifying which elements of the microarray bind to the patient's nucleic acid sample and which do not. The image is then analyzed to determine which specific nucleic acid fragments were present in the original sample and an assessment is then made as to whether a particular disease, mutation or other condition is present in the patient sample.
By way of specific example, a particular element of the microarray can be exposed to fluorescently-labeled fragments of DNA representative of a particular type of cancer. If that element of the array fluoresces under fluorescent illumination, it is known that the DNA of the sample contains the DNA sequence indicative of that particular type of cancer. Hence, a conclusion can be drawn that the patient providing the sample is already afflicted with that particular type of cancer or, alternatively, is possibly predisposed towards that cancer. By providing a wide variety of known DNA fragments on the microarray, the resulting fluorescent image can be analyzed to identify a wide range of conditions.
The detection of interactions on solid surfaces has been used for a variety of applications, including the identification of infectious organisms in clinical specimens (Spargo et al., (1993), Mol. Cell. Probe 7: 395-404; Martin, (1994) in The Polymerase Chain Reaction (Mullis, Ferre & Gibbs, eds.), pp. 406-17. Berkhauser, Boston), the quantitation of mRNA for gene expression analysis (Schena et al., (1995) Science 270: 467-70), and the sequencing or resequencing of genomic DNA on high-density “chip” arrays (Chee et al., (1996) Science 274: 610-13).
As noted, one embodiment of DNA microarray technology involves the attachment of a fluorescent label to a probe nucleic acid sequence, which is allowed to hybridize with a DNA sequence bound to a surface. Duplex formation is detected after removing the unhybridized DNA from the solid surface. Detection of fluorescently emitted photons is required to indicate the formation of a hybridization duplex and, therefore, analysis of high-density arrays labeled in this manner can require high-resolution fluorescence microscopes. Alternatively, indirect detection of hybridization can be accomplished using sandwich assays where the surface-bound hybrid is subsequently hybridized to an additional signal probe that carries one or more fluorescent labels or enzymes that impart fluorescent capability to a non-fluorescent substrate. Spargo et al., (1993) Mol. Cell. Probe 7: 395-404. In another embodiment, melting profiles can be examined in lieu of the more common fluorescent approach (Taton et al., (2000) Science 289:1757-1760).
The use of DNA hybridization arrays (“DNA microarrays”) has also had an impact on the technology available for sequencing cDNA, for sequencing mRNA and for determining the expression levels of selected genes. Stewart, (2000) Genome Res. 10:1-3; Yershov et al., (1996) Proc. Natl. Acad. Sci. U.S.A. 93:4913-4918; Lockhart et al., (1996) Nature Biotechnol. 14: 1675-1680; Ferguson et al., (1996) Nature Biotechnol. 14: 1680-1684. While this technology does not necessarily compete with current DNA sequencing methodology, it is very useful for rapid determination of the DNA content of mRNA expression level in a cell. Bulyk et al., (1999) Nat. Biotechnol. 6: 573-7; Lockhart & Winzeler, (2000) Nature 405: 827-36; Eisen & Brown, (1999) Method Enzymol. 303: 179-205. Reverse transcription of mRNA in reaction mixtures that comprise fluorescently labeled nucleotide triphosphates (NTPS) provides cDNA oligomers that can subsequently be hybridized to a surface. Thus, these microarrays are useful for genomic analysis in the laboratory and are of increasing importance in medical applications. Halushka et al., (1999) Nat. Genet. 22: 239-47; Rockett & Dix, (1999) Environ. Health Persp. 107: 681-85.
Efficient and rapid detection of mRNA expression levels has become particularly relevant, because mRNA analysis can allow a researcher or clinician to arrive at a conclusion or diagnosis based on an understanding of observed changes in protein expression. While DNA microarray technology is currently used predominantly as a tool for research, it has the potential to play a valuable role as a diagnostic tool.
The currently available detection strategy for DNA hybridization on surface arrays employs fluorescently labeled oligonucleotides and a reader consisting of a fluorescence microscope. Lipschutz et al., (1999) Nature Genet 21: 20-24. Currently, the most compact array is the GeneChip™ array that consists of 65,536 single stranded DNA sequences on a chip. Lipschutz et al., (1999) Nat. Genet. Sup. 21: 20-24; Harrington et al., (2000) Curr. Opin. Microbiol. 3: 285-91. Hybridization is probed by determining the fluorescence intensity at the location of each individual sequence of the DNA array. In the ideal case only complementary DNA in solution will hybridize and produce a fluorescent signal. However, non-specific binding and single/multi-base mismatches can provide a significant background signal. Although the background intensity is often weaker for non-specific interactions, the interpretation of the hybridization assay in terms of sequence becomes much more difficult. Consequently, statistical analysis is often required to extract the appropriate sequence information.
Protein and antibody arrays have also been developed that employ similar principles, but do not employ photocurrent-based detection methods. In protein and antibody arrays, binding of an analyte permits detection of a target protein. Relative quantities of material or binding constants can also be determined depending on the experimental configuration. However, in these methods detection of binding is usually based a fluorescence measurement. This requires fluorescent labeling of the target analyte, which can be undesirable.
A number of technological barriers have inhibited the employment of microchip and microarray technology in routine diagnostic and other applications. For example, the labeling of probe and/or target DNA with fluorescent moieties requires a cumbersome procedure and expensive detection apparatus is required for the detection of the labeled probe. Additionally, problems with false positive signals typically associated with the read-out of chip-based hybridization assay have been an obstacle. Moreover, hardware requirements have limited the use of microarrays useful for screening large numbers of samples. These and other problems are solved in whole or in part by the present invention.